UTS:SCIENCE Metabolic

Biochemistry

Lecture 3: Part 2
Enzyme Kinetics

Dr Evelyne Deplazes

Lecture notes adapted from material

by Dr Samiar Aili
 Metabolic
Learning outcomes Biochemistry

• Understand and describe the role of enzymes if

biochemical reactions
• Understand and read equilibrium diagrams
(concentration vs time plots) for reactions in the
presence and absence of enzymes ie the effect of
enzymes on the kinetics of reactions
• Understand the effect of of enzymes on the process
of the reactions and equilibrium
• Read free energy diagrams and understand the effect
of enzymes on the free energy of the reaction
 Metabolic
Learning outcomes Biochemistry

• Understand the concept of a enzyme binding site

• Understand the difference between rate constants
and equilibrium constants
• Understand the relationship between velocity and
rate constants
• Describe the different sections in a product vs time
plot with respect to steady state enzyme kinetics
 Metabolic
Learning outcomes Biochemistry

• Being able to produce and read Michaelis-Menten

plots
• Being able to produce and read Lineweaver-Burke
plots
• Estimate Vmax and Km from Michaelis-Menten plots
• Calculate Vmax and Km from Lineweaver-Burke plots
 Metabolic
Enzymes Biochemistry

• Catalyse reactions – increase the rate of

a reaction without themselves being
changed
• Recyclable and highly specific
• All enzymes are proteins, but not all
proteins are enzymes
 Metabolic
Remember equilibrium? Biochemistry

Start Reaction:
Products
Concentration (mmol/L)

A + B à C+ D
Reactants As time Passes:

Start
A+B↔ C+ D
Time (sec)
reaction
After about 60 secs the concentrations of reactants and products does not
change.
EQUILIBRIUM has been REACHED: forward and reverse rates are equal
concentrations will stay equal until the equilibrium is disturbed.
 Metabolic
Equilibrium with enzymes Biochemistry
Concentration (mmol/L)

Start Reaction:

A + B à C+ D
As time Passes:

A+B↔ C+ D
Time (sec)

With enzyme (dotted): Without enzyme:

Equilibrium reached Equilibrium reached
at 30 seconds at 60 seconds

7
 Metabolic
Equilibrium with enzymes Biochemistry

• With enzymes, the point of equilibrium is reached

faster.
• This does NOT mean that there is more products, it
just means that we got the products quicker.
• Therefore, enzyme speed up the rate of reaction, but
they do NOT alter the equilibrium end-point
• Enzyme lower the activation energy but do NOT
change the ∆G
 Metabolic
No change in free energy Biochemistry

9
 Metabolic
Enzymes lower activation energies Biochemistry

10
 Enzyme Specificity Metabolic
Biochemistry

• Enzymes are specific i.e. an enzyme

catalyses a very specific reaction
• Specificity is related to:
• Size and shape
• Electrostatic repulsion and attraction
• Hydrophobicity and hydrophilicity

11
Stereospecificity of Enzymes Metabolic
Biochemistry

• Isomers – molecules with the same chemical formula

but different spatial arrangement of the atoms

• Enantiomers are mirror-image molecules that are

structurally equivalent but cannot be superimposed
because of their "handedness," or chirality.

• Enzymes are also stereospecific

12
 Metabolic
Thalidomide Biochemistry

• Thalidomide: drug used to ease

morning sickness in the late 50s+ Therapeutic Teratogenic
• Shortly after, ~10,000 babies born
with phocomelia worldwide
– Only 50% survived

13
 Metabolic
Classification of Enzymes Biochemistry

Oxidoreductases
catalyse oxidation-reduction reactions BH2 + A à B + AH2

Transferases
catalyse transfer of functional groups from one
molecule to another D-B + A-H à D-H + A-B

Hydrolases
catalyse hydrolytic cleavage A-B + H2O àA-H + B-OH
Ligases and Synthetases
Bond formation (reverse of hydrolase) coupled to ATP hydrolysis

Lyases
catalyse removal of a group from or addition of a group to a double bond or other
cleavages involving electron rearrangement A-B à A + B
[ synthases: A + B àA-B ]
Isomerases
catalyse intramolecular rearrangement R-A-B à A-B-R
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 Metabolic
Rate constants ≠ Equilibrium constant Biochemistry

Rate constant (k) Equilibrium constant (Keq)

• Rate of a reaction • Ratio of concentration of

• the rate of conversion of products to concentration
reactants into products of reactants.
• measured in moles per sec
products

k K
k1, k-1 Keq
kf, kr
 Metabolic
Reaction Rate Biochemistry

A B
 Metabolic
Reaction rate (k) Biochemistry

A B
k

• A is the reactant
• B is the product -[A] = k[B] decrease in [A]
• k = the rate constant = [B] = k[A] increase in [B]
the rate of conversion of
reactants into products
[A] denotes concentration of A
[B] denotes concentration of B
 Metabolic
Forward and reverse reaction Biochemistry

A A B
Forward reaction
B
Reverse reaction

k +1 k-1

k+1 is also called kf k-1 is also called kr

Forward reaction Reverse reaction

• A is the reactant • B is the reactant
• B is the product • A is the product

-[A] = k[B] decrease in [A] [A] = k[B] increase in [A]

[B] = k[A] increase in [B] -[B] = k[A] decrease in [B]
 Metabolic
Reaction Rate - Velocity Biochemistry

• V = k.[S]
– V: Velocity (rate of reaction)
– k: rate constant
– [S]: substrate concentration

• Rate is the amount of substrate converted per unit of

time.
• Remember substrate can either be a product or
reactant depending on the direction of the
equilibrium
 Metabolic
At Equilibrium k+1 = k-1 Biochemistry

Equilibrium

k +1 z
k -1

V (forward) = V (reverse)

• At equilibrium: Vforward = Vreverse

• Can be written as: k+1[S]forward = k-1[S]reverse

• Reaction for AàB

– k+1[A] forward = k-1[B] reverse
 Metabolic
Relationship between Keq and k Biochemistry

• Rearranging the following:

k+1[A] forward = k-1[B] reverse

k+1 = ! 𝐟𝐨𝐫𝐰𝐚𝐫𝐝
k−1 " 𝐫𝐞𝐯𝐞𝐫𝐬𝐞
["]
• Equilibrium constant Keq= [$]

k
𝑲𝒆𝒒 = +1 = [𝐁]
k−1 [𝐀]
 Metabolic
Two-step enzyme reactions Biochemistry

Step 1 Step 2

Enzyme
Enzyme + Enzyme +
substrate
substrate product
Step 1 complex Step 2

Enzyme + k +1 Enzyme k +2 Enzyme +

substrate substrate product
k -1 complex k -2
 Metabolic
Two-step enzyme reactions Biochemistry

Step 1 Step 2 kcat

Enzyme + k +1 Enzyme k +2 Enzyme +
substrate substrate product
k -1 complex
k -2

• Kcat: number of substrate molecules converted to a

product over a specified time for one enzyme
• No reverse reaction (k-2)=0
• k2= rate limiting step
• Concentration of enzyme substrate complex is
constant i.e. steady state kinetics
 Metabolic
Steady state enzyme kinetics Biochemistry

• Steady state: state of reaction where concentration of

enzyme-substrate complex is constant
– Linear section of graph
– Lag phase: enzyme and substrate are docking, no product made
– Linear phase: steady state, same concentration of product made over
time
– Plateau phase: steady state lost, no more substrate available to make
product
Plateau: Equilibrium reached

Linear: steady state, increase in [Product] linear with time

Lag phase
 Metabolic
Michaelis-Menten equation Biochemistry

• Leonor Michaelis and Maud Menten developed an alternate

graph to describe enzyme kinetics for a single substrate reaction

Vmax [S]
V0 =
• Where:
K m + [S]
– V0= Initial velocity
– Vmax= Maximum velocity
– [S]= Substrate concentration
– Km= Michaelis constant
 Metabolic
Michaelis-Menten (MM) Plot Biochemistry

• Plot of reaction velocity vs substrate concentration axis

• Vmax= Maximum velocity

– Maximum velocity that the
reaction reaches
• Km= Michaelis-Menten
constant
– The substrate concentration
at half Vmax
• The MM equation is only
relevant when the plot shows a
hyperbolic relationship
between [S] and V0
 Metabolic
Maximal Velocity (Vmax) Biochemistry

• Vmax is the rate of the reaction

when substrate molecules
completely fill (saturate) the
enzyme's active sites

• Reflects how fast the enzyme can

catalyze the reaction.

• Given by the asymptote to the

velocity curve as the substrate
concentration is extrapolated to
infinity.
 Metabolic
Different perspective Biochemistry

• Left and middle image are linear section in graph

• Plateau is due to too much substrate and not
enough enzymes

[S] [S] [S]

• Active sites vacant • Active sites vacant • Active sites occupied

• Enzyme can work faster • Enzyme can work • Enzyme cannot work
• Rate can increase faster faster
• Rate can increase • Rate cannot increase
 Metabolic
Acetaldehyde Dehydrogenase Biochemistry

Alcohol
Dehydrogenase
CH3CH2OH + NAD+ CH3CHO + NADH + H+
(ethanol) • Oxidation of ethanol (acetaldehyde)
• Reduction of NAD+

+ H 2O
CH3CHO + NAD+ CH3COO- + NADH + 2H+
(acetaldehyde) Acetaldehyde (acetate)
Dehydrogenase

• TOXIC
2 forms:
• Hangovers • High Km (cytosol)
• Low Km (mitochondria)
 Metabolic
High vs low Km Biochemistry

Cytosol Mitochondria
High Km vs Low Km

• Slower reaction rate • Faster reaction rate

• Higher concentration of substrate • Lower concentration of substrate
required to start conversion required to start conversion
• Reaches half Vmax much slower • Reaches half Vmax faster
 Metabolic
Michaelis-Menten plot Biochemistry

Vmax (160 (μmol/L) / min)

160
V ((μmol/L) / min)

140 Vmax (150 (μmol/L) / min)

120
100 Mitochondria
80 Cytosol
60
40
20
0
0 50 100 150 200 250
Km Km [S] (μmol/L)
(30 μmol/L) (120 μmol/L)

à Non-linear plot, therefore too hard to read, estimates only

 Metabolic
Lineweaver-Burk equation Biochemistry

• Double-reciprocal equation
– Both sides of equation are inverted
– 1 divided by the Michaelis-Menten equation

Vmax [S]
V0 =
K m + [S]

1 Km 1 1
Lineweaver-
Burk Plot = +
V0 Vmax [S] Vmax
Straight line
equation
y = m x + b
 Metabolic
Lineweaver-Burk Plot Biochemistry

1 Km 1 1
= +
V0 Vmax [S] Vmax

y = m x + b

• To calculate Km and Vmax

– Need both X and Y
X intercepts
!"
• X-intercept=
#!
Y – Rearrange to get:
!"
– Km=
X−intercept
"
• Y-intercept=
$!"#
– Rearrange to get:
!"
– Vmax=
Y−intercept
 Metabolic
Questions for learning Biochemistry

Which of the following statements about enzymes is

correct.
a) Speed up reactions so they go to completion
b) Speed up reactions so all reactants are used
c) Speed up reactions so equilibrium is reached faster
than an uncatalysed reaction
d) Speed up reactions to stop them reaching
equilibrium
e) None of the above

34
 Metabolic
Questions for learning Biochemistry

Which of the following statements about enzymes is

correct.
a) Speed up reactions so they go to completion
b) Speed up reactions so all reactants are used
c) Speed up reactions so equilibrium is reached faster
than an uncatalysed reaction
d) Speed up reactions to stop them reaching
equilibrium
e) None of the above

35
 Metabolic
Questions for learning Biochemistry

Which of the following statements about enzymes is

correct. Enzymes are ________
a) Carbohydrates
b) Fats
c) Lipids
d) RNA
e) Proteins

36
 Metabolic
Questions for learning Biochemistry

Which of the following statements about enzymes is

correct. Enzymes are ________
a) Carbohydrates
b) Fats
c) Lipids
d) RNA
e) Proteins

37
 Metabolic
Question Biochemistry

What is reaction rate?

a) Amount of reactant converted into product in a specific time
period
b) Time taken to create a specific amount of product in a
reaction
c) Time taken to for a concentration of reactants to be used
during a reaction
d) The time taken for a reaction to reach equilibrium
 Metabolic
Answer Biochemistry

What is reaction rate?

a) Amount of reactant converted into product in a specific
time period
b) Time taken to create a specific amount of product in a
reaction
c) Time taken to for a concentration of reactants to be used
during a reaction
d) The time taken for a reaction to reach equilibrium
 Metabolic
Question Biochemistry

In the equation ‘V = k.[S]’, what does the ‘k’ stand for?

a) Equilibrium constant of the reaction
b) Rate constant of the reaction
c) Temperature of the reaction
d) Sum of the kinetics in the reaction
 Metabolic
Answer Biochemistry

In the equation ‘V = k.[S]’, what does the ‘K’ stand for?

a) Equilibrium constant of the reaction
b) Rate constant of the reaction
c) Temperature of the reaction
d) Sum of the kinetics in the reaction
 Metabolic
Question Biochemistry

Enzyme steady state kinetics is when:

a) The reaction is at equilibrium
b) No further product is being made
c) Substrate is being consumed at maximal rate
d) The enzyme-substrate concentration appears constant

Autumn 2014 Copyright UTS 2014 42

 Metabolic
Answer Biochemistry

Enzyme steady state kinetics is when:

a) The reaction is at equilibrium
b) No further product is being made
c) Substrate is being consumed at maximal rate
d) The enzyme-substrate concentration appears constant

Autumn 2014 Copyright UTS 2014 43

 Metabolic
Question Biochemistry

The Michaelis-Menten plot is

a) A graph that compares substrate concentration to reaction
rate
b) A graph from which maximal reaction rate can be estimated
c) A graph from which the Michaelis-Menten constant can be
determined
d) All of the above
e) None of the above

Autumn 2014 Copyright UTS 2014 44

 Metabolic
Question Biochemistry

• The table on the next page shows data for the enzyme reaction
that catalyses the conversion of reactant A to product P
• For each mol of A, 1 mol of P is produced
• In a spreadsheet, plot the data given in the table. Make sure to
label your axes
• Answer the following questions
– When does the reaction reach equilibrium?
– Which part of the plot do you use to calculate the activity of the enzyme?
– Use your plot to fit a trend line to the steady-state part of the plot
– Use your plot to calculate the activity of the enzyme
 Metabolic
Biochemistry

Time (min) Concentration of Product P

(µmol/mL)
0 0
1 4.5
2 9
3 13.5
4 18
5 22
6 24
7 25
8 26
9 26.5
10 26.9
 Metabolic
Answer Biochemistry

The Michaelis-Menten plot is

a) A graph that compares substrate concentration to
reaction rate
b) A graph from which maximal reaction rate can be
estimated
c) A graph from which the Michaelis-Menten constant can
be determined
d) All of the above
e) None of the above

Autumn 2014 Copyright UTS 2014 47

 Metabolic
Question Biochemistry

An enzyme with a high Km:

a) Needs a lot of substrate present before ½ maximal velocity is
reached
b) Needs a small amount of substrate present before ½
maximal velocity is reached
c) Needs a lot of substrate present before maximal velocity is
reached
d) Needs a small amount of substrate present before maximal
velocity is reached

Autumn 2014 Copyright UTS 2014 48

 Metabolic
Answer Biochemistry

An enzyme with a high Km:

a) Needs a lot of substrate present before ½ maximal
velocity is reached
b) Needs a small amount of substrate present before ½
maximal velocity is reached
c) Needs a lot of substrate present before maximal velocity
is reached
d) Needs a small amount of substrate present before
maximal velocity is reached

Autumn 2014 Copyright UTS 2014 49

 Metabolic
Questions for learning Biochemistry

The image on the next slide shows concentration vs time graphs for a
reaction where reactant A is converted into product B. One graph is for the
reaction in the absence of a catalytic enzyme, the other graph is for
the reaction in the presence of a catalytic enzyme. Which of the following
statements is correct about these graphs
a) In the graph on the right, the enzymes reduces the amount of product
produced in the reaction
b) The graph on the left shows the reaction without enzymes because
products are produced at a slower rate
c) The enzyme does not change the rate at which reactants are consumed
d) The graph on the right shows the reaction without enzymes because
reactants are consumed at a faster rate
 Metabolic
Questions for learning Biochemistry
 Metabolic
Questions for learning Biochemistry

The image on the next slide shows concentration vs time graphs for a
reaction where reactant A is converted into product B. One graph is for the
reaction in the absence of a catalytic enzyme, the other graph is for
the reaction in the presence of a catalytic enzyme. Which of the following
statements is correct about these graphs
a) In the graph on the right, the enzymes reduces the amount of product
produced in the reaction
b) The graph on the left shows the reaction without enzymes because
products are produced at a slower rate
c) The enzyme does not change the rate at which reactants are consumed
d) The graph on the right shows the reaction without enzymes because
reactants are consumed at a faster rate
 Metabolic
Questions for learning Biochemistry

Which of the following statements about enzymes is

correct. Enzymes ________
a) change an unfavourable reaction into an favourable
reaction
b) convert a exergonic reaction into an endergonic
recitation
c) Lower the change in Gibbs free energy (∆G)
d) Increase the activation energy of a reaction
e) Lower the activation energy of a reaction

53
 Metabolic
Questions for learning Biochemistry

Which of the following statements about enzymes is

correct. Enzymes ________
a) change an unfavourable reaction into an favourable
reaction
b) convert a exergonic reaction into an endergonic
recitation
c) Lower the change in Gibbs free energy (∆G)
d) Increase the activation energy of a reaction
e) Lower the activation energy of a reaction

54
 Metabolic
Questions for learning Biochemistry

Which of the following statements about enzymes is

NOT correct.
a) Enzymes alter the concentrations of products at the
equilibrium end-point
b) With enzymes, the point of equilibrium is reached
faster.
c) Enzymes speed up the rate of reaction
d) Enzyme lower the activation energy

55
 Metabolic
Questions for learning Biochemistry

Which of the following statements about enzymes is

NOT correct.
a) Enzymes alter the concentrations of products at
the equilibrium end-point
b) With enzymes, the point of equilibrium is reached
faster.
c) Enzymes speed up the rate of reaction
d) Enzyme lower the activation energy

56